Serological and molecular screening of umbilical cord blood for Toxoplasma gondii infection.

BACKGROUND: Human umbilical cord blood has proven to be a successful alternate source of hematopoietic stem cells for pediatric patients with major hematologic disorders. Toxoplasma gondii is a global opportunistic protozoan which cause fatal complications in immunocompromised individuals. AIM: Our goal is to study the prevalence of toxoplasmosis in umbilical cord blood (UCB) and to assess the sensitivity of ELISA and PCR for Toxoplasma infection screening. MATERIAL AND METHODS: One hundred cord blood samples were collected immediately after delivery. Anti-Toxoplasma IgG and IgM antibodies were determined using ELISA method; Toxoplasma DNA was detected using nested PCR technique. Total nucleated cells (TNC) and HB were also determined. Demographic data and risk factors data related to the transmission of toxoplasmosis, were collected from mothers. RESULTS: Among 100 cord blood samples, 36 (36%) were positive for anti-Toxoplasma IgG antibodies and 6 (6%) were positive for anti-Toxoplasma IgM antibodies. The nested PCR showed 11 (11%) samples containing Toxoplasma DNA from which, 6 (55%) samples were IgM positive. There was no significant association between the risk of Toxoplasma transmission and cord blood positivity for toxoplasmosis. CONCLUSION: Owing to the prevalence of toxoplasmosis, its rapid progression and its fatal outcome in immunocompromised patients, cord blood screening for toxoplasmosis with nested PCR should be incorporated into cord blood bank screening protocols.

Genetic polymorphisms of IL-23R (rs7517847) and LEP (rs7799039) among Egyptian patients with hepatocellular carcinoma.

BACKGROUND: Genetic polymorphisms of IL-23 R (rs7517847) and LEP (rs7799039) have been stated to be associated with various types of human cancers. The purpose of this work is to test the association of these genetic polymorphisms with hepatocellular carcinoma (HCC) among Egyptian patients. SUBJECTS AND METHODS: This study involved 150 unrelated Egyptian HCC patients in addition to 100 healthy controls from the same locality. DNA was genotyped for these genetic polymorphisms using the PCR-RFLP technique. RESULTS: The frequency of the IL-23 R (rs7517847) G and LEP (rs7799039) G alleles were significantly higher among HCC patients compared to controls (p = .004 and .02). However, HCC patients with the IL-23 R GG and LEP GG genotypes showed no significant difference compared to others regarding their clinical and laboratory markers. CONCLUSIONS: IL-23 R (rs7517847) and LEP (rs7799039) polymorphisms were associated with an increased risk but not affecting the clinical presentation of HCC among Egyptian patients.

Genetic polymorphisms of NFκB1-94ins/delATTG and NFκBIA-881A/G genes in Egyptian patients with colorectal cancer.

To assess the association of genetic polymorphisms of NFκB1 and NFκBIA genes with the susceptibility to colorectal cancer (CRC). Subjects included 100 Egyptian patients with CRC (60 males and 40 females) in addition to 85 healthy controls (47 males and 38 females) from the same locality. For all participants, genetic polymorphisms of NFκB1-94ins/delATTG (rs28362491) and NFκBIA-881A/G (rs3138053) were detected by using restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). CRC patients showed a significantly higher frequency of the NFκB1-94ins/ins genotype than controls (30 vs. 4.7%) that was significant in the recessive (OR 17.69, 95% CI 5.41-57.82, p < 0.0001) and codominant models (OR 18.28, 95% CI 4.87-68.6, p < 0.0001). The NFκB1-94ins allele frequency was significantly higher among patients than controls (58 vs. 39%, OR 2.18, 95% CI 1.4-3.3, p = 0.0004). We also noticed that the genotype G/G of NFκBIA-881 polymorphism was present in patients (4%) while it was absent (0%) in controls with increased frequency of the NFκBIA-881G allele in patients compared to controls (23 vs. 14%, p = 0.041). These polymorphisms were more associated with smoking and advanced tumor staging. This study indicates that the NFκB1-94ins/ins genotype was associated with the risk of developing colorectal cancer in Egyptian subjects. Also, CRC cases showed an increase in the frequency of NFκBIA-881G allele but not reaching statistical significance for multiple comparisons.

Association of ACE and MTHFR genetic polymorphisms with type 2 diabetes mellitus: Susceptibility and complications.

HYPOTHESIS/INTRODUCTION: Polymorphisms of angiotensin converting enzyme (ACE) and methylene-tetrahydrofolate reductase (MTHFR) genes have been proposed to be associated with type 2 diabetes mellitus (T2DM) with conflicting results. This work was planned in order to check for the association of these polymorphisms with the susceptibility for and complications of T2DM among Egyptian cases. MATERIALS AND METHODS: This is a case controlled study involving 203 patients with T2DM and 311 healthy controls. Polymorphic variants of ACE I>D and MTHFR (677 C>T and 1298 A>C) were determined using the polymerase chain reaction (PCR) restriction analysis technique. RESULTS: The susceptibility to T2DM was higher among subjects having the MTHFR 677TT (odds ratio (OR)=2.2, p=0.01), MTHFR 1298 AA (OR=1.84, p=0.001) and ACE (ID+II) (OR=2.0, p=0.0007) genotypes. Logistic regression analysis showed that MTHFR 677T allele was a risk factor for diabetic retinopathy (DR) (OR=3.47, p<0.001), diabetic polyneuropathy (DPN) (OR=5.2, p<0.0001) and ischemic heart disease (IHD) (OR=2.9, p<0.05), while MTHFR 1298 C allele was a risk factor for DR (OR=4.2, p<0.001) and the ACE DD genotype was a risk factor for DPN (OR=3.1, p<0.001). CONCLUSIONS: The MTHFR 677 TT genotype was associated with T2DM susceptibility and complications (DR, DPN and IHD). The MTHFR 1298 CC, AC and ACE DD genotypes were associated with DR and DPN.

Cure rate of Helicobacter pylori infection in Egyptian children related to CYP2C19 gene polymorphism.

OBJECTIVES: This study was done in order to investigate the effect of CYP2C19 genetic polymorphism on the cure rate of children who received proton pump inhibitors (PPI)-based triple therapy for treating Helicobacter pylori (H. pylori) infection. METHODS: Participants included 100 children with H. pylori-positive gastritis diagnosed by endoscopy and biopsy in addition to H. pylori stool antigen test. Cure rate was assessed after 1 month of completion of a triple treatment course for 14 days. CYP2C19 polymorphism was analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Results showed that cases with a CYP2C19 genotypic status consistent with the heterozygote extensive metabolizers (HetEMs) had a higher cure rate of H. pylori when compared with the homozygote extensive metabolizers (HomEMs) although it was statistically nonsignificant (84.6 vs. 69.2). In addition, the poor metabolizers (PMs) had a higher cure rate compared with those of the HomEMs which was also statistically nonsignificant (77.8 vs. 69.2). The cure rate was also higher among both the groups of HetEMs and PMs combined together compared to the HomEMs (OR = 2.15, p > 0.05). Comparing cases regarding their age, gender, and severity of H. pylori gastritis revealed a better cure rate in the age group >10 years, in females and in mild and moderate cases than other cases although statistically nonsignificant. CONCLUSION: The higher cure rate of H. pylori infection using the triple therapy for 2 weeks among HetEMs and PMs cases compared to the HomEMs might warrant a need for a therapy augmentation or modification for the HomEMs.

Tumor necrosis factor-α gene polymorphism relationship to seminal variables in infertile men.

OBJECTIVE: To assess the tumor necrosis factor (TNF)-α gene polymorphism relationship with seminal variables in fertile men (N) and those with asthenozoospermia (A), asthenoteratozoospermia (AT), and oligoasthenoteratozoospermia (OAT). MATERIALS AND METHODS: A total of 50 infertile men without a female factor who were attending a fertility clinic and 48 fertile men were randomly screened for semen analysis, analysis of the TNF-α promoter region for polymorphism, seminal caspase-9, acrosin activity, α-glucosidase, and reproductive hormones. RESULTS: The TNF-α GG genotype was present in 83.9%, 72.7%, 66.7%, and 59.5%, the TNF-α AA genotype in 3.2%, 6.8%, 10.4%, and 11.9%, and TNF-α AG genotype in 12.9%, 20.5%, 22.9%, and 28.6% in the N, A, AT, OAT groups, respectively. The occurrence of A allele was significantly greater among infertile patients than among fertile controls (21.6% vs 9.7%; odds ratio 0.388, 95% confidence interval 0.2 to 0.75, P = .005). Men with the TNF-α AA genotype demonstrated a significant decrease in the sperm count, sperm motility, normal sperm morphology, acrosin activity, and seminal α-glucosidase and a significant increase in seminal caspase-9 compared with those with the TNF-α GG genotype. CONCLUSION: This single nucleotide polymorphism in the TNF-α(-308) gene was associated with significantly increased seminal caspase-9 and a significantly decreased sperm count, sperm motility, normal sperm morphology, acrosin activity, and seminal α-glucosidase.

MTHFR C677T, A1298C and ACE I/D polymorphisms as risk factors for diabetic nephropathy among type 2 diabetic patients.

BACKGROUND: Genetic variations have been proposed to play a role in the susceptibility to diabetic nephropathy. OBJECTIVES: To check for the association of genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and angiotensin converting enzyme (ACE) genes with the development of diabetic nephropathy among type 2 diabetic patients. METHODS: Participants comprised 202 patients with type 2 diabetes, of whom 102 were affected with diabetic nephropathy. Genetic variants corresponding to MTHFR C677T, A1298C and ACE I/D genotypes were determined using the PCR technique coupled with digestion and restriction analysis. RESULTS: Cases with diabetic nephropathy had a significantly higher frequency of the MTHFR 677 TT, 677 CT, ACE DD mutant genotypes compared with diabetic cases without nephropathy. Analysis of the association of studied MTHFR C677T, A1298C and ACE I/D polymorphisms with albuminuria showed that the MTHFR 677 T polymorphism, in the recessive and dominant models, was a risk factor for both micro and macroalbuminuria, while the ACE DD mutant genotype was a risk factor for microalbuminuria and the MTHFR 1298C in the dominant model only was a risk factor for macroalbuminuria. CONCLUSION: These findings indicate that ACE and MTHFR genetic polymorphisms might be considered as genetic risk factors for diabetic nephropathy among patients with type 2 diabetes.

Association of cytokine gene polymorphisms with psoriasis in cases from the nile delta of egypt.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease with an immunogenetic background. This work was planned to check for the association of polymorphisms related to cytokine genes TNF-α(-308) (G/A), IL-10(-1082) (G/A), IL-6(-174) (G/C), and IL-1Ra (VNTR) with psoriasis in cases from Egypt. MATERIALS AND METHODS: This work included 46 cases with psoriasis recruited from the Dermatology Departments, University Hospitals, Nile Delta region of Egypt. They included 14 males and 32 females with an age mean ± SD of 46.68 ± 12.16 years and range of 15-70 years. Their genotypes were compared to 98 healthy controls of matched age and sex from the same locality. Genotyping was done through deoxyribonucleic acid amplification using PCR with sequence specific primers for polymorphic alleles. RESULTS: Compared to controls, cases showed significant higher frequency of certain genotypes including IL-6(-174) CC (P < 0.001, OR = 6.7), IL-10(-1082) GG (P < 0.05, OR = 5.1), and TNF-α(-308) GG (P < 0.05, OR = 3.7). TNF-α(-308) GG and IL-10(-1082) GG genotypes were higher among cases with plaque subtype of moderate severity. Combined heterozygosity for IL-10 GA, IL-6 GC with TNF GA showed a significant low frequency among studied cases. CONCLUSION: Genetic polymorphisms related to IL6, IL10, and TNF-α genes showed a particular pattern of association with psoriasis that may have a potential impact on disease counseling and management.

Association of cytokine gene polymorphisms with psoriasis in cases from the Nile Delta of Egypt.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease with an immunogenetic background. This study was planned to check for the association of polymorphisms related to cytokine genes TNF-alpha-308(G/A), IL-10-1082(G/A), IL-6-174(G/C), and IL-1Ra (VNTR) with psoriasis in cases from Egypt. METHODS: This study included 46 cases with psoriasis recruited from the Department of Dermatology, University Hospitals, Nile Delta region of Egypt. They included 14 males and 32 females with a mean age +/- SD of 46.68 +/- 12.16 years and a range of 15 to 70 years. Their genotypes were compared to 98 healthy controls of matched age and sex from the same locality. Genotyping was done through DNA amplification using PCR with sequence-specific primers for polymorphic alleles. RESULTS: Compared to controls, cases showed a significantly higher frequency of certain genotypes including IL-6-174 CC (p < 0.001, OR = 6.7), IL-10-1082 GG (p < 0.05, OR = 5.1), and TNF-alpha-308 GG (p < 0.05, OR = 3.7). Combined heterozygosity for IL-10 GA, IL-6 GC, and TNF GA showed a significant low frequency among the cases studied. CONCLUSION: Genetic polymorphisms related to the IL6, IL10, and TNF-alpha genes showed a particular pattern of association with psoriasis that may have a potential impact on disease counseling and management.

Gene polymorphisms of TNF-alpha-308 (G/A), IL-10(-1082) (G/A), IL-6(-174) (G/C) and IL-1Ra (VNTR) in Egyptian cases with type 1 diabetes mellitus.

BACKGROUND: Type 1 diabetes (T1D) is a genetically conditioned autoimmune disease in which cytokines play an important role. Objectives. To check for the association of polymorphisms of cytokine genes with type 1 diabetes. Subjects. This work included 50 cases with T1D and 98 healthy individuals from the Nile Delta region of Egypt. Cases included 20 males and 30 females with a median age of 25 and range of 15-50 years. METHODS: DNA was amplified using PCR with sequence-specific primers for detection of polymorphisms related to tumor necrosis factor (TNF)-alpha(- 308) (G/A), interleukin (IL)-10(- 1082) (G/A), IL-6(- 174) (G/C), and IL-1Ra (VNTR). RESULTS: Cases with T1D showed significant higher frequency of genotypes of TNF-alpha(- 308) AA (p < 0.001, odds ratio (OR) = 7.91), IL-6-17CC (p < 0.05, OR = 3.36) and IL-1Ra A1A1 (p < 0.05, OR = 3.68) with significant lower frequencies of TNF-alpha(- 308) GA, and IL-1Ra A1A2 genotypes (p < 0.001 and < 0.05, respectively). They also showed significant higher frequency of TNF-alpha(- 308) allele A (p < 0.05, OR = 2.0), IL-1Ra allele A1 (p < 0.05, OR = 2.98) with a significant lower frequency of TNF-alpha(- 308) G allele and IL-1Ra A2 allele (p < 0.05). No significant difference was detected among cases in relation to IL-10(- 1082) (G/A) genotypes or alleles nor in relation to age, sex, consanguinity or family history of the disease. CONCLUSIONS: Polymorphisms related to TNF-alpha and IL-1Ra genes may be considered genetic markers for T1D among Egyptians with a potential impact on family counseling and management.

Frequency of factor V Leiden mutation in Egyptian cases with myocardial infarction.

BACKGROUND: Acute myocardial infarction (MI) is death or necrosis of myocardial cells due to lack of blood supply. One of the causes may be thrombosis resulting from inherited resistance to activated protein C caused by the factor V Leiden (FVL) mutation. OBJECTIVES: To check for the presence of FVL mutation among Egyptian cases with MI compared to normal population controls. SUBJECTS AND METHODS: This study is a form of a prospective controlled study including 44 MI cases with an age ranging from 25 to 80 years and sex of 36 males (81.8%) and 8 females (18.2%). These cases were taken randomly from those admitted in the Intensive Care Units of Mansoura University Hospitals, Egypt. Of these cases, 10 cases (55.6%) were smokers, 7 cases (75.0%) had a positive family history of MI, 8 cases (18.18%) were diabetic and 20 cases (45.45%) were hyperlipidemic. For association and risk analysis, cases were compared to 211 healthy unrelated control subjects of matched age and sex. Factor V Leiden (G1691A) gene mutation was detected using a multiplex allele-specific PCR amplification. RESULTS: Mutant A allele frequency of factor V Leiden was significantly higher in cases (30.68%) than in controls (10.19%) (p<0.0001, OR=3.9). Total cases showed significant higher frequency heterozygous mutant genotype GA (43.0%) compared to controls (16.6%), (p<0.0001, OR=4.45). Also total cases showed significant higher frequency of the homozygous mutant genotype AA (9.0%) compared to controls (1.9%), (p=0.0094, OR=8.19). On the other hand, no significant difference was found between cases subgroups related to age, sex, smoking, diabetes and hyperlipedemia. CONCLUSION: Frequency of factor V Leiden mutation among Egyptian cases with myocardial infarction is relatively high. So, families of affected subjects should be genotyped and counseled for proper prophylaxis.

Gene polymorphisms of tumor necrosis factor alpha-308 and interleukin-10-1082 among asthmatic Egyptian children.

Tumor necrosis factor (TNF) alpha-308 and interleukin (IL)-10(-1082) have potent inflammatory responses in the process of airway inflammation in asthma. The purpose of this study was to check for association of polymorphisms related to cytokine genes with susceptibility and severity of bronchial asthma in Egyptian children. Blood samples of 69 asthmatic children receiving treatment and follow-up at the Allergy and Respiratory Medicine Unit, Mansoura University Children Hospital, Mansoura, Egypt, were subjected to DNA extraction and amplification using polymerase chain reaction with sequence-specific primers for detection of single nucleotide polymorphisms in the promoter regions of cytokine genes TNF-alpha(-308(G-->A)), IL-10(-1082(G-->A)). Compared with normal controls, Egyptian asthmatic children showed a significant higher frequency of IL-10(-1082) G/G homozygosity genotype (p < 0.001; odds ratio [OR] = 7) with lower frequency of G/A heterozygosity genotype among cases. This finding also was detected in cases with persistent asthma and eczema. These cases showed significant lower frequency of TNF-alpha-308 G/A heterozygosity (p < 0.05; OR = 0.44). Also, male cases, cases with positive family history, and those patients with persistent types of asthma showed a higher frequency of TNF-alpha-308 G/G homozygosity. IL-10(-1082(G-->A)) G/G and TNF-alpha-308(G-->A) G/G may be a contributing factor in susceptibility as well as severity of asthma among Egyptian children. Separate studies should be specified relating these cytokine genotypes to response to various modalities in asthma therapy. This study reports that IL-10(-1082(G-->A)) G/G and TNF-alpha-308(G-->A) G/G genotypes may be contributing factors in susceptibility as well as in severity of asthma among Egyptian children. Separate studies may be specified relating these cytokine genotypes to response to various modalities in asthma therapy.

Clinical and molecular diagnosis of Familial Mediterranean Fever in Egyptian children.

BACKGROUND: Familial Mediterranean Fever (FMF) is an autosomal inherited disorder affecting certain races including Arabs. Diagnosis depends mainly on clinical basis, but mild forms may remain undiagnosed. OBJECTIVES: This study aims at an accurate diagnosis of FMF in Egyptian children by detection of genetic mutations in addition to clinical assessment. SUBJECTS AND METHODS: Subjects included 66 Egyptian cases (37 males and 29 females) with a mean age of onset of 6.9 years. They had been referred from health centers and hospitals of the Delta region, Egypt. Analysis of the clinical manifestations was performed using Tel-Hashomer criteria in addition to 10 items clinical score system. For all these cases, DNA analysis was made for three common mutations M680I, M694V, and V726A using amplification refractory mutation system (ARMS-PCR) technique. RESULTS: Most of the cases had attacks ranging from 3-5 days duration with the mean of 3.6 days. Their rate of recurrence was variable but 47 % of them had suffered attacks 10-30 times/year. Abdominal pain was the most common symptom (87.9%) followed by fever (82%), arthritis or arthralgia (56.1%), chest pain (45%) and myalgia (6%). Laparotomy had been done during attacks for exploration or appen-dectomy in 27.7% of cases. Positive mutations were detected among 42 cases (63.6%), of them 14 (21.2%) were compound heterozygotes, 7 (10.6%) were had homozygotes while 21 (31.8%) were simple heterozygotes. Allele M694V was the most frequent one (18.8%) followed by V726A (17.4%) and M680I (12.1%). Taking positive mutation as a guide for diagnosis, a cutoff clinical score level was determined with =15 for unlikely, =20 for definite and 15-20 for probable diagnosis. CONCLUSION: Diagnosis of FMF among Egyptian children cases although based mainly on clinical suspicion requires to be confirmed through detection of the corresponding mutation which can be easily made using the simple ARMS-PCR technique.

Diagnosis of Sex Chromosome Disorders and Prenatal Diagnosis of Down Syndrome using Interphase Fluorescent In-Situ Hyperidization Technique.

BACKGROUND: Thousands of infants are born each year with chromosomal abnormalities that severely impact physical and mental development. Among common genetic disorders are Down syndrome (trisomy 21) and sex chromosomal disorders. OBJECTIVES: Evaluation of guidelines used for prenatal diagnosis of Down syndrome (DS) as well as sex chromosomal disorders including interphase Fluorescent In Situ Hyperidization (FISH) technique. METHODS: Enrolled cases were among those presenting to Genetics and Neonatology Units, Mansoura and Ain-Shams University hospitals,(Egypt) during 2002 to 2004. These included: Groups 1 comprised fifty pregnant women presenting for genetic counseling. They were subjected to complete history analysis, ultrasound examination in addition to triple screening test (for alpha feto protein (AFP), human chorionic goandotrophin (HCG) and unconjugated esteriol (E2). Results were confirmed by doing routine karyogram on cultured amniotic fluid. Groups 2 comprised suspected cases with sex chromosomal disorders including neonates with ambiguous genitalia (64 cases) and adults with primary amenorrhea (69 cases) or infertility (38 cases). They were subjected to a diagnostic workup including RESULTS: Among the pregnant women group, seven were found to be at a high risk of having DS fetuses including 3 cases with a history of affected off-springs, 2 cases with age above 35 years, and 2 cases with high triple test. Only one case had positive trisomy 21 on interphase FISH confirmed by karyogram on cultured amniotic cells. The other 6 ladies had normal FISH confirmed by karyograms. Regarding the other group, 5 cases out of the 9 females were proved to be feminized males, one proved mosaic turner, one proved mixed gonadal dysgenesis and 2 normal females. On the other hand one out of three males were proved to be verilized female while the other one was a male with incomplete testicular feminization and the last one was a male with infertility diagnosed as Klinefelter syndrome at the age of 26 years. CONCLUSION: Interphase FISH is a rapid, accurate and very sensitive method in sex chromosom and autosomal abnormalities. It adds to the diagnostic utility of routine cytogenetics and its use on interphase nuclei overcomes the difficulty of conventional cytogenetics. It could be used in the prenatal diagnosis of DS in addition to ultrasonography, and triple test.

Molecular analysis of A1AT (S and Z) and HFE (C282Y and H63D) gene mutations in Egyptian cases with HCV liver cirrhosis.

BACKGROUND: Alpha-1-antitrypsin (A1AT) S and Z deficiency alleles and hemochromatosis (HFE) mutant C282Y, H63D alleles were reported to potentially affect the liver even if present in a heterozygous state. OBJECTIVES: This is a cross-sectional, randomized, case controlled study for evaluation of the frequency of these alleles in Egyptian patients with HCV liver cirrhosis and of their association with the disease. SUBJECTS: This study included 48 cases with viral C cirrhosis recruited from the Hepatology Unit, Mansoura University Hospital, Egypt, and 70 unrelated healthy controls. METHODS: PCR amplification of relevant gene segment followed by restriction enzyme digestion Taq1 for detection of A1AT gene S and Z alleles, digestion with Rsa I and Bcl I for HFE gene C282Y and H63D alleles. These alleles were then characterized through analysis of resulting restriction fragment length polymorphism (RFLP). RESULTS: Both heterozygous (MS) and homozygous (SS) genotypes were significantly more frequent in cases than in controls ( P<0.05, RR= 2.23 and 2.17 respectively). Gene frequency of S allele was higher in cases than controls (P<0.05, RR=2.17). Homozygosity (ZZ) genotype, present only in cases (6.3% vs 0.0% in controls,) did not reach statistical significance. HFE gene heterozygosity for H63D allele was detected in 20.0% of cases and 21.4% of controls, whereas C282Y allele was detected neither among cases nor in controls. CONCLUSION: The presence of the relatively high frequency of A1AT S and HFE H63D allele carriers in Egyptian cases of HCV liver cirrhosis suggest the necessity to implement routine molecular analysis of these genes for detection of risk genotypes among affected families.